One of the most remarkable characteristics of the immune system is its ability to generate a large and diverse repertoire of antibody molecules that specifically recognize the native form of epitope(s) of potential pathological consequence in an attempt to protect the organism. Such a diversity that protects an organism from a variety of infectious pathogens and toxic substances has been exploited to produce high affinity receptors/antibody for use in immunodiagnostics, molecular probes and immunotherapy. For some applications, it is necessary to reduce antibody size to improve biodistribution, and reduce immunogenicity and side reactions arising from antibody effector functions. The Fv, consisting of associated VH and VL domains, constitutes the minimum functional antigen-binding fragment of a conventional antibody. Because of the inherent instability of VH–VL Fv heterodimers, single domain antibody, i.e., VH only, is expected to be stable because of its small size. This may be particularly so as homodimers of heavy (H)-chain alone occur naturally as antibody in camels. The murine VHs, unlike camel, are ‘sticky’ because of exposure of a large hydrophobic surface spanning 500–800 A0. In addition to poor solubility behaviour the murine VHs have an average CDR3H length of 9 amino acids in contrast to human (12 residues) and camels (14 residues). Thus, the potential antigen-binding surface of murine VH is smaller as compared to human antibodies of potential therapeutic significance. A Fv or single domain antibody with a longer CDR3H is, therefore, of potential interest from an antibody engineering perspective as it would potentially provide large antigen surface binding area.
The variable-region of immunoglobulin (Ig) bear V-region epitope capable of stimulating both humoral and cellular immune responses upon hetero-, allo or iso-immunization (Nisonoff, A. and Lamoyi, E. 1981. Implication of the presence of an internal image of the antigen in anti-idiotypic antibodies: possible application to vaccine production. Clin. Imm. Immunopath. 21:397–406; Zaghouani, H. et al. 1993. Presentation of a viral T cell epitope expressed in the CDR3 region of a self-immunoglobulin molecule. Science 259:224–227; Zaghouani, H. et al. 1993. Engineered immunoglobulin molecules as vehicles for T cell epitope. Intern. Rev. Immunol. 10:265–278). The V-region epitope of an Ig often correlates with the amino acid composition of the CDR regions. Since the CDR loops vary both in composition and size, these provide permissive sites for foreign (e.g., microbial origin) epitope insertion that would create an antigenized Ig of desired epitope. Prior research indeed demonstrates the ability to manipulate the entire CDRS of an immunoglobulin for generation of functional chimeric antibodies of interest (Morisson, Science 229:1202; Jones et al.1986. Nature 321:522; Verhoeyen et al. 1988. Science. 239:1534; Riechmann et al. 1988. Nature 332:323). Antigenization of immunoglobulin is, however, hindered by shorter CDRs in immunoglobulins from mice and man that permit incorporation of T cell epitopes (linear) but not B cell epitopes (conformational) for use as vaccine vectors.
The inventors observed that approximately 9% of VDJ rearrangements encode functional bovine IgM antibody with a CDR3H length up to 61 amino acids with multiple cysteine residues (Saini, 1999). Such an exceptionally long CDR3H is generated by unusually long germline D-genes with unique composition that exist in cattle (Farbod Shojaei, MSc Thesis (2001). Molecular aspects of bovine DH genes. University of Guelph, Guelph) and is the first ever to be documented in a species and is of significant interest to exploit its potential in engineering antibodies, either as Fv or single domain, of diagnostic and therapeutic significance. In addition to the long CDR3H, the configurational diversity from such a long primary sequence with multiple even numbered cysteine residues is expected to contribute to the immunoglobulin fold capable of binding to an infinite array of chemical structures. In addition, bovine germline D genes provide molecular genetic marker for cattle breeding strategies and forensic analysis.